Compositions and methods for detecting allogeneic matter in a subject

ABSTRACT

The present disclosure provides a panel of nucleic acid molecule primers specific for HLA-specific alleles and other genetic polymorphisms, which are useful for quantitatively amplifying these markers to detect, diagnose, and monitor individuals who have or are at risk of certain disease conditions, such as autoimmune disease, proliferative disease, infectious disease, allograft rejection, or pregnancy-related pathologies.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under A1041721,A1045659, AR048084, A1045952, and HL117737 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 411C2_SEQUENCE_LISTING_V2.txt. The text file is16.5 KB, was created on Feb. 17, 2023.

BACKGROUND 1. Technical Field

The present disclosure relates to compositions and methods forquantitatively amplifying nucleic acid molecules from biological samplesand, more particularly, to a panel of nucleic acid molecules useful forquantitative amplification of target markers for detecting, diagnosing,or monitoring individuals who have or are at risk of certain diseaseconditions, such as autoimmune disease, proliferative disease,infectious disease, allograft rejection, or pregnancy-relatedpathologies.

2. Description of Related Art

Individuals harbor small amounts of foreign cells or DNA, referred to asmicrochimerism (“Mc”). Acquisition of Mc occurs naturally duringpregnancy through transplacental cell trafficking between mother andfetus, although Mc may be acquired through other sources (e.g., bloodtransfusion, organ transplantation). An adult woman may have acquired Mcfrom her own mother (maternal Mc, “MMc”) while she herself was a fetus.This “graft,” acquired during fetal immune system development, canremain in her system into adulthood (Maloney et al., J. Clin. Invest.104:41, 1999; Lambert et al., Arth. Rheu. 50:906, 2004) and represents apre-existing inhabitant as she experiences pregnancy herself Duringsubsequent pregnancies, new fetal sources of microchimerism (fetal Mc,“FMc”) can be acquired and also remain for years. The interactions ofeach of these grafts with the host, and with other pre-existinginhabitants, may be beneficial or detrimental to an individual.

Disease and Mc may have a functional connection since persistence of Mchas been shown to be associated both positively and negatively withcertain disease states, such as autoimmune disease (Evans et al., Blood93:2033, 1999; Yan et al., Arth. Rheu. 63:640, 2011), malignancy (Gadi,Breast Cancer Res. Treat. 121:241, 2010; Gadi et al., PLoS ONE 3:e1706,2008), and transplant rejection (Gadi et al., Clin. Chem. 52:379, 2006;Gadi et al., Transpl. 92:607, 2011). In autoimmunity, higher detectionrates and concentrations of Mc suggest a possible allo-autoimmune orauto-alloimmune functionality (Gammill and Nelson, Int. J. Dev. Biol.54:531, 2010). In the case of malignancy, lower detection rates andconcentrations of Mc in cancer cases suggest a possiblegraft-versus-tumor effect (Gadi, Cancer Lett. 276:8, 2009). Intransplantation, the presence of Mc may serve as a biomarker formonitoring allograft survival (Gadi et al., 2006, 2011).

From the foregoing, a need is apparent for improved compositions andmethods for sensitively and quantitatively identifying allogeneic cells,tissues, and nucleic acids resulting from medical conditions orinterventions.

BRIEF SUMMARY

In one aspect, the present disclosure provides compositions comprising aforward nucleic acid molecule, a reverse nucleic acid molecule, and aprobe nucleic acid molecule comprising a fluorophore and a quencher,wherein each individual composition is specific for HLA-B*44,HLA-DRB1*01, HLA-DRB1*15/16, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*07,HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*10, HLA-DRB1*14, HLA-DRB4,HLA-DQA1*01, HLA-DQA1*03, HLA-DQA1*05, HLA-DQB1*02, HLA-DQB 1*03,HLA-DQB1*04, HLA-DQB 1*06, SE-HR, SE-LR, GSTT 1, AT3, or Tg, as setforth in Table 1, and wherein each nucleic acid molecule has a lengthranging from about 10 nucleotides to about 35 nucleotides.

In another aspect, the present disclosure provides methods forquantitating microchimerism, monitoring allograft rejection,anti-malignancy effects, engraftment dominance, or the like, wherein themethods involve amplifying nucleic acid molecules of a target biologicalsample using one or more specific nucleic acid compositions listed inTable 1 and analyzing for the presence or absence of certain specificamplified markers in the target biological sample to monitor or assessmicrochimerism, monitoring allograft rejection, anti-malignancy effects,engraftment dominance, or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of the longitudinal assessment of donor DNAconcentrations and acute transplant rejection (ACR). High concentrationsof donor DNA (given in qEq/106 host genomes), as measured with theDQB1*06 nucleic acid molecule composition in Q-PCR, were detected inserum from a patient at points correlating with ACR in pancreas andkidney specimens (*) or pancreas alone (**). Day 196 was negative forACR in the pancreas.

FIG. 2 shows a bar graph illustrating quantitative expression ofmaternal microchimerism in different tissues, peripheral bloodmononuclear cells (PBMCs), and bone marrow (B. marrow) from a probandwith systemic sclerosis. Tp=autologous hematopoietic stem celltransplantation.

DETAILED DESCRIPTION

The present disclosure provides compositions of nucleic acid moleculesfor use in quantitatively detecting the presence of allogeneic material,such as cells or DNA, in a subject. Such compositions and methods can beuseful in detecting microchimerism, transplant rejection, malignancyrelapse, potential pathology associated with pregnancy, infectiousdisease, or the like. Exemplary compositions include a forward nucleicacid molecule, a reverse nucleic acid molecule, and a probe nucleic acidmolecule comprising a fluorophore and a quencher, wherein eachcomposition is specific for a particular HLA allele, for particularpolymorphisms of an HLA allele, or for other genetic polymorphisms withdifferent alleles or a null allele.

By way of background, some cells may (and often times do) trafficbetween a mother and fetus during pregnancy. Surprisingly, small numbersof these allogeneic cells can persist in their respective hosts decadeslater. Microchimerism (Mc) refers to an individual harboring a smallnumber of cells, or DNA, derived from another individual. As notedabove, the instant disclosure provides compositions and methods forexamining a wide breadth of consequences of naturally-acquired Mc acrossall of human health (Adams and Nelson, JAMA 291:1127, 2004).

For example, Mc may have an effect on or have a role in autoimmunedisease. In a first study of Mc, elevated levels of fetal Mc in bloodwere found in women with scleroderma compared to healthy women (Nelsonet al., Lancet 351:559, 1998). Fetal Mc (FMc) has since beeninvestigated in primary biliary cirrhosis, thyroiditis, Sjögren'ssyndrome, polymorphic eruption of pregnancy and rheumatoid arthritis.Maternal Mc (MMc) can be found in her adult progeny (Maloney et al., J.Clin. Invest. 104:41, 1999). MMc has been studied in neonatal lupus,systemic lupus and myositis. In neonatal lupus with heart block,maternal cells in the heart were cardiac myocytes (Stevens et al.,Lancet 362:1617, 2003). Thus, microchimeric cells could be targets oralternatively could help repair damaged tissues. FMc may be beneficialduring pregnancy in women with rheumatoid arthritis as elevated levels,assessed by Q-PCR, significantly correlated with pregnancy-inducedamelioration of arthritis (Yan et al., Arthritis Rheum. 54:2069, 2006).

In cancer, hematopoietic cell transplantation (HCT) donor cells providean advantage against recurrent leukemia and other malignancies. Byanalogy, FMc might be responsible for protection from breast cancerobserved in women who have had prior pregnancies compared to those whohave not. In other types of malignancies, anecdotal reports indicatethat microchimeric cells may cause or result in malignancy.

For infectious disease, T lymphocytes are an important determinant ofimmune reactions between one's own cells and foreign cells. Humanimmunodeficiency virus (HIV) and acquired immunodeficiency syndrome(AIDS) are characterized by critical deficiencies in CD4+ T cells. MMccan be examined in HIV and AIDS by employing HLA and other geneticpolymorphism specific Q-PCR to quantify MMc in men with HIV to correlateresults progression as compared to non-progression to AIDS.

In the field of transplantation, iatrogenic chimerism can result. But,donor Mc may facilitate graft acceptance. Until recently, donor Mc wasmeasured as male DNA in female recipients, but it is now clear thatwomen commonly have male DNA from prior pregnancies. Thus, theapplication of specific Q-PCR assays of this disclosure provides a majorstep forward for these studies. In hematopoietic cell transplantation(HCT), graft-versus-host disease (GVHD) occurs more often if the donoris a woman with prior pregnancies. Female apheresis products were foundto contain male Mc, consistent with the idea that fetal Mc contributesto GVHD (Adams et al., Blood 15:3845, 2003). In kidney, pancreas andislet transplantation embodiment, the panel of Q-PCR assays of theinstant disclosure are useful for testing serial serum samples, whichprovides a non-invasive test for early rejection (see Example 1). TheQ-PCR assays of the instant disclosure may also be used to monitor thefate of co-transplanted hematopoietic cells as it affects a kidneyallograft from the same donor.

In the context of pregnancy, the compositions and methods of the instantdisclosure are useful for identifying changes in the maternal carriageof fetal cells throughout the course of normal pregnancy (see Example2). In certain embodiments, the tools and methods of the instantdisclosure can be used to examine pathologic conditions associated withpregnancy (pre-eclampsia, early fetal loss, and abnormally geneticfetuses, as examples) and their association with deviations from normalpregnancy patterns.

Prior to setting forth this disclosure in more detail, it may be helpfulto an understanding thereof to provide definitions of certain terms tobe used herein. Additional definitions are set forth throughout thisdisclosure.

In the present description, the terms “about” and “consistingessentially of” mean±20% of the indicated range, value, or structure,unless otherwise indicated. It should be understood that the terms “a”and “an” as used herein refer to “one or more” of the enumeratedcomponents. The use of the alternative (e.g., “or”) should be understoodto mean either one, both, or any combination thereof of thealternatives. As used herein, the terms “include,” “have” and “comprise”are used synonymously, which terms and variants thereof are intended tobe construed as non-limiting.

As used herein, “fluorophore” refers to a molecule that emits light of acertain wavelength after having first absorbed light of a specific, butshorter, wavelength, wherein the emission wavelength is always higherthan the absorption wavelength.

As used herein, “quencher” refers to a molecule that accepts energy froma fluorophore in the form of light at a particular wavelength anddissipates this energy either in the form of heat (e.g., proximalquenching) or light of a higher wavelength than emitted from thefluorophore (e.g., FRET quenching). Quenchers generally have a quenchingcapacity throughout their absorption spectrum, but they perform bestclose to their absorption maximum. For example, Deep Dark Quencher IIabsorbs over a large range of the visible spectrum and, consequently,efficiently quenches most of the commonly used fluorophores, especiallythose emitting at higher wavelengths (like the Cy® dyes). Similarly, theBlack Hole Quencher family covers a large range of wavelengths (over theentire visible spectrum and into the near-IR). In contrast, Deep DarkQuencher I and Eclipse® Dark Quencher effectively quench the lowerwavelength dyes, such as FAM, but do not quench very effectively thosedyes that emit at high wavelengths.

As used herein, “target nucleic acid molecules” and variants thereofrefer to a plurality of nucleic acid molecules that may be found incertain biological samples but may be missing from others, depending onthe genetic make-up of the subject and the extent of microchimerismpresent. Nucleic acid molecules include those from natural samples(e.g., a genome, RNA), or the target nucleic acid molecules may besynthetic samples (e.g., cDNA), recombinant samples, or a combinationthereof.

As used herein, a “nucleic acid molecule primer” or “primer” andvariants thereof refers to short nucleic acid sequences that a DNApolymerase can use to begin synthesizing a complementary DNA strand ofthe molecule bound by the primer. A primer sequence can vary in lengthfrom 5 nucleotides to about 50 nucleotides in length, from about 10nucleotides to about 35 nucleotides, and preferably are about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,or 35 nucleotides in length. In certain embodiments, a nucleic acidmolecule primer that is complementary to a target nucleic acid ofinterest can be used to initiate an amplification reaction, a sequencingreaction, or both.

For example, for quantitative PCR, the combination of an upstream orforward primer (5′) and a downstream or reverse primer (3′)complementary to a target sequence of interest (e.g., HLA-B, HLA-DRB)can be used to prime an amplification reaction to obtain the sequence ofthe nucleic acid molecule of interest. A “probe” oligonucleotidesequence is similar to a primer except that it will further contain afluorophore molecule and a quencher molecule and hybridize to a targetnucleic acid molecule of interest somewhere between the forward andreverse primer binding sites.

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of thisdisclosure. However, upon reviewing this disclosure one skilled in theart will understand that the invention may be practiced without many ofthese details. In other instances, newly emerging amplificationtechnologies, as well as well-known or widely available specific probeamplification methods and tools (e.g., Taqman® probes, Locked NucleicAcid (LNA) probes, Molecular Beacon probes, Scorpions® primers,Hybridization probes, MGB probes, QuantiProbes, Resonsense probes,Light-up probes, HyBeacon® probes, LUX primers, Yin-yang probes,Amplifluor®), have not all been described in detail to avoidunnecessarily obscuring the descriptions of the embodiments of thepresent disclosure.

Various embodiments of the present disclosure are described for purposesof illustration, in the context of use with HLA-specific alleles andcertain other genomic polymorphisms. But, as those skilled in the artwill appreciate upon reviewing this disclosure, use with other targetnucleic acid molecules may also be suitable.

In certain embodiments, the present disclosure provides methods fordetecting, diagnosing, or monitoring the presence of allogeneic cells,tissues, or nucleic acid molecules that may be involved in or associatedwith a particular medical condition, such as autoimmune disease,neoplastic disorders, infectious disease, transplant rejection, andpathologies associated with pregnancy. In further embodiments, themethods of the instant disclosure are sensitive enough to detect onechimeric genome in 10⁵ host genomes or 10⁶ host genomes when amplifyingspecific target nucleic acid molecules in presence of many differentnucleic acid molecules.

In further embodiments, the compositions and methods of this instantdisclosure will be useful in detecting rare nucleic acid molecules orcells against a large background signal, such as when monitoring for Mcin autoimmune disease, infectious disease, malignancies, transplantsubjects, pregnancy, or forensics. Additional embodiments may be used toquantify target nucleic acid molecules that may be indicative ofresponse to therapy or may be useful in monitoring disease progressionor recurrence. In yet other embodiments, these compositions and methodsmay be useful in detecting or monitoring target nucleic acid moleculesafter or during chemotherapy, autoimmune therapy, infectious diseasetherapy, or treatments of complications to pregnancy.

Representative nucleic acid molecule compositions of the presentdisclosure may be specific for HLA alleles, such as HLA-B*44,HLA-DRB1*01, HLA-DRB1*15/16, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*07,HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*10, HLA-DRB1*14, HLA-DRB4,HLA-DQA1*01, HLA-DQA1*03, HLA-DQA1*05, HLA-DQB1*02, HLA-DQB1*03,HLA-DQB1*04, or HLA-DQB1*06 (see Table 1). Alternatively, nucleic acidmolecule compositions of the present disclosure may be specific forother genetic polymorphisms, such as SE-HR, SE-LR, GSTT1, AT3, or Tg(see Table 1). In certain embodiments, each nucleic acid molecule has alength ranging from about 10 nucleotides to about 35 nucleotides.

Selecting nucleic acid molecule compositions of the present disclosureis not routine because many of the sequences tested did not work due tocross-reactivity with other genes. For example, the DRB1*11 specificprimers and probes described in Example 1 did not work because multiplecross-reactivity did not allow for analysis of this marker. Alternativesequences for this marker also did not work. Similarly, DRB1*05 andDQB1*05 did not work, and a particular primer pair for DRB1*10 (seeHLA-DRB1*10-2 SEQ ID NOS.:28 and 29 of Table 1) also did not work, aswell as various other primers and probes (not shown) for the targetslisted in Table 1.

The probe nucleic acid molecules of the compositions of the presentdisclosure are preferably dual labeled oligonucleotides that include afluorophore (e.g., FAM, TET, HEX, Cy®3, Cy®3.5, Cy®5, Cy®5.5, TAMRA,Yakima Yellow®, ROX) and a quencher (e.g., Deep Dark Quencher I, DeepDark Quencher II, DabCYL, Eclipse® Dark Quencher, Black Hole Quencher(BHQ-0, BHQ-1, BHQ-2, BHQ-3, TAMRA). For example, the instant disclosureprovides HLA and other genetic polymorphism-specific quantitative PCR(Q-PCR) primers and probes for use in diagnostics, detection of medicalconditions, or monitoring medical conditions.

An exemplary panel of primers and probes useful in the compositions andmethods of the instant disclosure are provided in Table 1.

TABLE 1 qPCR Primers and Probes* Target Forward (5′-3′) Reverse (3′-5′)Probe (5′-3′) HLA-B*44 CCG CGG GTA TGA TCC AGG TAT CGG CTC AGACCA GGA (SEQ ID CTG CGG AGC G TCA CCC AGC NO.: 1) (SEQ ID NO.: 2)GCA A (SEQ ID NO.: 3) HLA-DRB1*01 CAC GTT TCT TGT GCT GTC GAATC CTC TTG GTT GGC AGC TTA AGT GCG CAC GG ATA GAT GCA T (SEQ ID NO.: 4)(SEQ ID NO.: 5) TCT TTC CAG CAA CC (SEQ ID NO.: 6) HLA-DRB1*15/16CGT TTC CTG TGG GCA CGG ACT CGT CCC ATT CAG CCT AA (SEQ CCT CCT GGTGAA GAA ATG ID NO.: 7) TAT (SEQ ID ACA CTC CCT C NO.: 8) (SEQ ID NO.: 9)HLA-DRB1*03 CCA CGT TTC TTG T GCA GTA GTT TT CTC CTC CTG GAG TAC TCT ACGGTC CAC CCG GTT ATG GAA TC (SEQ ID NO.: 10) A C (SEQ ID GTA TCT GTCNO.: 11) CAG GT (SEQ ID NO.: 12) HLA-DRB1*04 CGT TTC TTG GAG CG CAC GTACAC CCG CTC CAG GTT AAA CA CTC CTC TTG CGT CCC GTT (SEQ ID NO.: 13)GTG (SEQ ID GAA (SEQ ID NO.: 14) NO.: 15) HLA-DRB1*07 CGT TTC CTG TGGC CCC GTA GTT AAG TGT CAT CAG GGT AAG TA GTG TCT GCA TTC TTC AAC(SEQ ID NO.: 16) CAC (SEQ ID GGG ACG GAG C NO.: 17) (SEQ ID NO.: 18)HLA-DRB1*08 A CGT TTC TTG G TCT GCA GTA TAT AAC CAA GAG TAC TCT ACGGGT GTC CAC GAG GAG TAC GG (SEQ ID NO.: 19) CAG (SEQ ID GTG CGC TTCNO.: 20) GAC AG (SEQ ID NO.: 21) HLA-DRB1*09 G CAC GTT TCT C CCC GTA GTTT TCT CCT CTT TGA AGC AGG A GTG TCT GCA GGT TAT AGA (SEQ ID NO.: 22)CAC (SEQ ID TGC CTC TGT NO.: 23) GCA GAT (SEQ ID NO.: 24) HLA-DRB1*10-1GGT TGC TGG AAA GTG TCC ACC AGT ACG CGC GAC GCG (SEQ ID GCG GCA (SEQGCT ACG ACA NO.: 25) ID NO.: 26) GCG AC (SEQ ID NO.: 27)HLA-DRB1*10-2^(¶) CGG TTG CTG GAA GGT GTC CAC AGT ACG CGC AGA  AGC G (SEQ CGC GG A  A (SEQ GCT ACG ACA ID NO.: 28) ID NO.: 29)GCG AC (SEQ ID NO.: 27) HLA-DRB1*14 CGG CCT GCT GCG AAC CCC GTACCG CCT CCG GA A  C (SEQ ID GTT GTG TCT CTC CAG GAG GT NO.: 31)GCA A (SEQ ID (SEQ ID NO.: 33) NO.: 32) HLA-DRB4*01 CAG GCT AAG TGTCCT GGT ACT TA TCT GAT CAG GAG TGT CAT TTC CCC CCA GGT GTT CCA CACC (SEQ ID NO.: 34) CA (SEQ ID TCG CTC CGT NO.: 35) (SEQ ID NO.: 36)HLA-DQA1*01 C CTG GAG AGG AGC CAT GTT ACC TCC AAA AAG GAG ACT GCTCT CAG TGC TTT GCT GAA (SEQ ID NO.: 37) ACC (SEQ ID CTC AGG CCA CNO.: 38) (SEQ ID NO.: 39) HLA-DQA1*03 AA TTT GAT GGA GC AAA TTGA TCT GCG GAA GAC GAG GAG TTC CGG GTC AAA CAG AGG CAA TAT (SEQ IDTCT (SEQ ID CTG CCA (SEQ ID NO.: 40) NO.: 41) NO.: 42) HLA-DQA1*05TTG CAC TGA CAA TGG TAG CAG AAC TTG AAC ACA TCG CT A  TC CGG TAG AGTAGT CTG ATT AA (SEQ ID NO.: 43) TGG (SEQ ID (SEQ ID NO.: 45) NO.: 44)HLA-DQB* C GTG CGT CTT GTA CTC GGC AG CGT CAC CGC GTG AGC AGA AGGGC AGG CA CCG GAA CTC C (SEQ ID NO.: 46) (SEQ ID NO.: 47)(SEQ ID NO.: 48) HLA-DQB1*03 CGG AGC GCG TGC CGT GCG GAG AG GAC TTC CTTGTT A (SEQ ID CTC CAA CTG CTG GCT GTT NO.: 49) (SEQ ID NO.: 50)CCA GTA CTC G (SEQ ID NO.: 51) HLA-DQB1*04 TGC TAC TTC ACC CTA TTC CAGTCG GTT ATA AAC GGG A A C TAC TCG GCG GAT GTA TCT (SEQ ID NO.: 52)TCA A (SEQ ID GGT CAC ACC NO.: 53) CCG (SEQ ID NO.: 54) HLA-DQB1*06GAC GTG GGG GTG CTG CAA GAT TTC CTT CTG GCT TAC CGC (SEQ ID CCC GCG GAGTT CCA GTA NO.: 55) (SEQ ID NO.: 56) CTC GGC AT (SEQ ID NO.: 57)SE-HR (QRRAA)† CCA GAA GGA CCT GTG TCT GCA CGG CCC GCC CCT GGA GC (SEQGTA GGT GTC TCT (SEQ ID ID NO.: 58) CAC  A G (SEQ ID NO.: 60) NO.: 59)SE-HR (QKRAA)† CCA GAA GGA CCT GTG TCT GCA CGG CCC GCT CCT GGA GC (SEQGTA GGT GTC TCT (SEQ ID ID NO.: 61) CAC  A G (SEQ ID NO.: 63) NO.: 62)SE-LR (DERAA) CCA GAA GGA CAT GTG TCT GCA CGG CCC GCT CCT GGA AG (SEQGTA GGT GTC CGT (SEQ ID ID NO.: 64) CAC  A G (SEQ ID NO.: 66) NO.: 65)GSTT1 TTC CAG GAG GCC GGG CAT CAG AAG GCC AAG CAT GAG (SEQ IDCTT CTG CTT GAC TTC CCA NO.: 70) TAT G (SEQ ID CCT GCA (SEQ ID NO.: 71)NO.: 72) AT3-S CCT CTC TCC ATA GCT TTA TAG CTT GGT TCA AAG AAA ACT ATGAAA AGG AAA TAC CCA CCC AGA GA (SEQ ID AGG AGA GTA (SEQ ID NO.: 75)NO.: 73) TG (SEQ ID NO.: 74) AT3-L CCT CTC TCC ATA GGA TTT TTTCCC TCT ACC AAG AAA ACT ATG GTT TCT CGT TGT AAT TC (SEQ AGA GA (SEQ IDTAA CTA AAT ID NO.: 78) NO.: 76) CAG (SEQ ID NO.: 77) Tg-ICAC CTC CAC CAC CGC AGA GTA TCC TGG CCC CCA TAG AGA (SEQ CAT TGT GAGATG TTC CCA ID NO.: 79) GTT TTA G (SEQ AGC TCT (SEQ ID ID NO.: 80)NO.: 81) Tg-D GGT TAC GGT GTC AGT TCC AGC TCT CCA GCC AGA AAA CCT GAAAA CAC TTG TCT GTG TTA (SEQ ID NO.: 82) AAG ATG (SEQ ATG TGA GCC CID NO.: 83) (SEQ ID NO.: 84) *Nucleotides identified in bold andunderline are an artificial nucleotide mismatch to the native sequence.^(¶)This particular pair of forward/reverse primers, namedHLA-DRB1*10-2, did not work. †The SE-HR (Shared Epitope-High Risk) qPCRreactions will also include an inhibitor oligonucleotide as follows: ACATCC TGG AGC AGG CGC GG (SEQ ID NO.: 85).

In a test for MMc in a child, for example, a person of skill in the artmay decide to target HLA-DRB1* as an approach to selecting one or morenucleic acid molecule compositions from Table 1. The initial step wouldbe to conduct HLA-genotyping on the child, mother and father. Forexample, the HLA genotypes may be HLA-DRB1*01/HLA-DRB1*15 for themother, HLA-DRB1*03/HLA-DRB1*14 for the father, andHLA-DRB1*01/HLA-DRB1*03 for the child. Examination of the HLA-genotypingindicates that the non-shared HLA allele of the mother as compared tothe child is HLA-DRB1*03. So, DNA is extracted from the child andinterrogated for MMc employing the appropriate HLA-specific Q-PCR assayfrom among the panel of assays listed in Table 1—in this case, one woulduse a composition of three nucleic acid molecules that includes SEQ IDNOS.:10, 11, and 12. In addition, an irrelevant HLA-specific Q-PCR assaycan be included in testing the child as a negative control.

In certain embodiments, the instant disclosure provides a process forquantitating microchimerism, comprising: (a) amplifying nucleic acidmolecules of a target biological sample using one or more specificnucleic acid compositions from Table 1; (b) amplifying nucleic acidmolecules of a control biological sample using the one or more specificnucleic acid molecule compositions of step (a); (c) comparing the amountof amplified nucleic acid molecules with a reference to quantitate thespecific nucleic acid molecule markers in the samples; wherein thepresence of certain specific amplified markers in the target biologicalsample indicates the presence of microchimerism.

Umbilical cord blood and bone marrow transplants can be used to cure orslow the progression of many cancers originating in the bone marrow(e.g., leukemia, myeloma) or lymphatic system (e.g., lymphoma). Incertain embodiments, nucleic acid molecule compositions and methods ofthe instant disclosure can be used to determine donor MMc to identifyone or more donor cord blood or donor bone marrow that will provide thegreatest anti-malignancy or anti-relapse potential.

For example, if the HLA-B* is being targeted, then HLA-genotyping wouldbe conducted on the patient, the patient's mother, a first cord blooddonor, the first cord blood donor's mother, a second cord blood donor,and the second cord blood donor's mother. The HLA genotypes may beHLA-B*08/HLA-B*15 for the patient, HLA-B*08/HLA-B*44 for the patient'smother, HLA-B*22/HLA-B*15 for the first cord blood donor,HLA-B*22/HLA-B*35 for the first cord blood donor's mother,HLA-B*08/HLA-B*13 for the second cord blood donor, and HLA-B*08/HLA-B*37for the second cord blood donor's mother. Examination of theHLA-genotyping indicates that the first cord blood donor shares thepatient's inherited paternal antigen (IPA), HLA-B*15; therefore, thefirst cord blood donor would provide the greatest anti-malignancy oranti-relapse potential. By way of illustration and not wishing to bebound by theory, the benefit for the patient to receive the firstdonor's cord blood would be due to effector T cells of the donor'smother that have previously been exposed to the same HLA molecule as thepatient's IPA (i.e., HLA-B*15). In contrast, effector T cells of themother donor for the second cord blood donor would have reactivity toHLA-B*13, which is not shared with the patient.

If, however, the mother does not have a unique HLA allele (i.e., themother and child are HLA identical or the child is HLA homozygous) or aset nucleic acid molecules of the instant disclosure are not available,testing can be done on genetic polymorphisms found on other chromosomes.Exemplary nucleic acid molecule compositions from Table 1 include, forexample, glutathione S-transferase 01 (GSTT1), anti-thrombin III long(AT3-L), anti-thrombin III short (AT3-S), thyroglobulin insertion (Tg-I)and thyroglobulin deletion (Tg-D). All nucleic acid moleculecompositions have been validated for specificity using Q-PCR assaystested against an extensive panel of DNA derived from fully HLAcharacterized cell lines from the 13th International HLA Workshop (seewww.ihwg.org; see also Marsh et al., Tissue Antigens 75:291, 2010).

In certain medical situations, patients may receive a double cord bloodtransplant. So in the previous example, although a patient may receive adouble cord blood transplant, only one of the donors may provide abenefit. In certain embodiments, the nucleic acid molecule compositionsand methods of the instant disclosure can be used to determine whichplurality of donor cord blood or donor bone marrow would be best tocombine. In a further embodiment, the nucleic acid molecule compositionsand methods of the instant disclosure can be used to determine whichdonor cord blood or donor bone marrow from a pooled transplant woulddominantly engraft.

For example, if the HLA-B* is again being targeted, then HLA-genotypingwould be conducted on the patient, the patient's mother, a first cordblood donor, the first cord blood donor's mother, a second cord blooddonor, and the second cord blood donor's mother. The HLA genotypes maybe HLA-B*08/HLA-B*15 for the patient, HLA-B*08/HLA-B*44 for thepatient's mother, HLA-B*22/HLA-B*12 for the first cord blood donor,HLA-B*22/HLA-B*35 for the first cord blood donor's mother,HLA-B*18/HLA-B*44 for the second cord blood donor, and HLA-B*18/HLA-B*37for the second cord blood donor's mother. Examination of theHLA-genotyping indicates that the second cord blood donor shares thepatient's non-inherited maternal antigen (NIMA), HLA-B*44; therefore,the second cord blood donor would provide the greatest potential fordominant engraftment—in this case, one would use a composition of threenucleic acid molecules that includes SEQ ID NOS.:1, 2, and 3.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DRB1*01, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:4, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:5, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:6.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DRB1*15/16, whereinthe forward nucleic acid molecule comprises a sequence as set forth inSEQ ID NO.:7, the reverse nucleic acid molecule comprises a sequence asset forth in SEQ ID NO.:8, and the probe nucleic acid molecule comprisesa sequence as set forth in SEQ ID NO.:9.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DRB1*03, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:10, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:11, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:12.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DRB1*07, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:16, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:17, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:18.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DRB1*08, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:19, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:20, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:21.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DRB1*09, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:22, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:23, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:24.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DRB1*10, wherein (a)the forward nucleic acid molecule comprises a sequence as set forth inSEQ ID NO.:25, the reverse nucleic acid molecule comprises a sequence asset forth in SEQ ID NO.:26, and the probe nucleic acid moleculecomprises a sequence as set forth in SEQ ID NO.:27.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DRB1*14, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:31, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:32, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:33.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DRB4, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:34, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:35, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:36.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DQA1*01, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:37, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:38, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:39.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DQA1*03, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:40, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:41, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:42.

In further embodiments, the instant disclosure provides a compositioncomprising comprises nucleic acid molecules specific for HLA-DQA1*05,wherein the forward nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:43, the reverse nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:44, and the probe nucleic acidmolecule comprises a sequence as set forth in SEQ ID NO.:45.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DQB1*03, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:49, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:50, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:51.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DQB1*04, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:52, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:53, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:54.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for HLA-DQB1*06, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:55, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:56, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:57.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for SE-HR, wherein (a) theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:58, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:59, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:60, or (b) the forward nucleic acidmolecule comprises a sequence as set forth in SEQ ID NO.:61, the reversenucleic acid molecule comprises a sequence as set forth in SEQ IDNO.:62, and the probe nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:63. In related embodiments, the aforementionedcompositions may further comprise an inhibitor nucleic acid moleculecomprising a sequence as set forth in SEQ ID NO.:85.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for SE-LR, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:64, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:65, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:66.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for GSTT1, wherein theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:70, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:71, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:72.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for AT3, wherein (a) theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:73, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:74, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:75, or (b) the forward nucleic acidmolecule comprises a sequence as set forth in SEQ ID NO.:76, the reversenucleic acid molecule comprises a sequence as set forth in SEQ IDNO.:77, and the probe nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:78.

In further embodiments, the instant disclosure provides a compositioncomprising nucleic acid molecules specific for Tg, wherein (a) theforward nucleic acid molecule comprises a sequence as set forth in SEQID NO.:79, the reverse nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:80, and the probe nucleic acid molecule comprises asequence as set forth in SEQ ID NO.:81, or (b) the forward nucleic acidmolecule comprises a sequence as set forth in SEQ ID NO.:82, the reversenucleic acid molecule comprises a sequence as set forth in SEQ IDNO.:83, and the probe nucleic acid molecule comprises a sequence as setforth in SEQ ID NO.:84.

In any of the aforementioned embodiments, the instant disclosureprovides a composition wherein the probe comprises a fluorophore at the5′-end and a quencher at the 3′-end. In any of the aforementionedembodiments, the instant disclosure provides a composition wherein thefluorophore is FAM and the quencher is TAMRA or BHQ. In any of theaforementioned embodiments, the instant disclosure provides acomposition wherein the probe further comprises a duplex stabilizer,such as at least one LNA or MGB.

Although specific embodiments and examples of this disclosure have beendescribed herein for illustrative purposes, various equivalentmodifications can be made without departing from the spirit and scope ofthe invention, as will be recognized by those skilled in the relevantart after reviewing the present disclosure. The various embodimentsdescribed can be combined to provide further embodiments. The describeddevices, systems and methods can omit some elements or acts, can addother elements or acts, or can combine the elements or execute the actsin a different manner or order than that illustrated, to achieve variousadvantages of the invention. These and other changes can be made to theinvention in light of the above detailed description.

In general, in the following claims, the terms used should not beconstrued to limit the invention to the specific embodiments disclosedin the specification. Accordingly, the invention is not limited by thedisclosure, but instead its scope is determined entirely by thefollowing claims

EXAMPLES Example 1 Measuring Allograft Rejection by HLA-Specific Q-PCRof Transplant Recipient Serum

There is no reliable serum marker available to monitor incipientpancreas or islet-cell rejection. Quantification of donor specificgenomic DNA in serum was measured as a marker of rejection. A panel ofHLA-specific quantitative PCR assays (Q-PCR) was used to test 158 serafrom 42 pancreas-kidney transplant recipients. The HLA-specific primersand probes used were directed to DRB1*01 (SEQ ID NOS:4, 5 and 6),DRB1*11 (forward primer CAG ACC ACG TTT CTT GGA GTA CTC TAC, SEQ IDNO.:86; reverse primer CCT TCT GGC TGT TCC AGT ACT CCT, SEQ ID NO.:87,and probe CGC TCC GTC CCA TTG AAG AAA TGA CA, SEQ ID NO.:88), DQA1*01(SEQ ID NOS:37, 38 and 39), DQA1*03 (SEQ ID NOS:40, 41 and 42), DQB1*02(SEQ ID NOS:46, 47 and 48), DQB1*03 (SEQ ID NOS:49, 50 and 51), DQB1*06(SEQ ID NOS:55, 56 and 57), DRB4*01 (SEQ ID NOS:34, 35 and 36), and B*44(SEQ ID NOS:1, 2 and 3). Temporally related biopsies for 65 serapermitted analysis for correlation of donor DNA concentrations withrejection.

Briefly, a calibration curve for the HLA-specific assay of interest wasgenerated with known quantities of genomic DNA [0, 0.5, 1, 5, 10, 50,100, and 500 genome-equivalents (gEq)] derived from Epstein-Barrvirus—transformed cell lines that were previously HLA typed and known tobe homozygous for the allele of interest. A separate β-globincalibration curve was created to quantify the total amount of genomicDNA derived from both host and donor within each specimen. Total genomicDNA was isolated from patient sera (200 to 500 μL) by use of a DNA MiniKit (Qiagen) with a final elution volume of 50 μL. Specimen HLA Q-PCRreactions contained 10 μL of template DNA (or 5 μL for the β-globinassay to maximize the amount of DNA eluate available for HLA assays), 25μL of TaqMan® Universal Master Mix (Applied Biosystems), 300 nM each ofthe forward/reverse primers (MWG Biotech), 100 nM dual labeled probe(MWG Biotech), and DNase/RNase-free water to a final volume of 50 μL. Ofnote, to prevent PCR competition for the reagents between the moreprevalent β-globin PCR product and the less prevalent HLA-specificproduct, assays were performed in a non-multiplexed format withHLA-specific and β-globin assays contained in separate wells on the sameplate. Four wells were measured, on average, for each serum sample forHLA and 1 well for β-globin. PCR reactions were incubated in an ABIPrism 7000 thermocycler for 2 min at 50° C., followed by 45 cycles of95° C. for 15 s and 60 to 64° C., depending on the HLA assay, for 1 min.HLA (or β-globin) quantities were determined for sample wells byplotting on the calibration curve the point at which a fluorescencethreshold for a given assay was exceeded. Results were rejected andassays repeated if either calibration curve correlation coefficient (r2)was <0.99.

HLA quantities were expressed as the total number of cell-free gEq/mL ofserum as calculated by the equation: ΣHLA Values/Σ Sample Volume×elutionvolume×1000/serum volume=gEq/mL. The equation accounts for theproportion of the DNA extract that was amplified for each target allele(elution volume/sum of sample volume). In addition, we determined theratio of donor DNA to host cell-free DNA (as determined by simultaneousβ-globin Q-PCR) in the serum to control for nonspecific increases intotal soluble DNA. The ratio was subsequently corrected to reflectwhether the assayed HLA allele was present in 1 or 2 copies in the donorgenome.

Results: Donor DNA concentrations were higher in sera from recipientswho had experienced allograft rejection (n=31) than from those who hadnot (n=34). Median concentrations, expressed as the genome-equivalent(gEq) number of donor cells per 10⁶ host cells, were 2613 and 59gEq/10⁶, respectively (P=0.03). Relative concentrations of donor DNA inhost serum (gEq/106 host genomes) over time for an exemplary patientprobed with DQB1*06 is shown in FIG. 1 .

Conclusion: Q-PCR for donor-specific genetic polymorphisms is anoninvasive approach to monitor pancreas-kidney, as well as other typesof allograft rejection.

Example 2 Measuring Maternal Microchimerism by HLA-Specific Q-PCR

Microchimerism (Mc), originating from bidirectional fetal-maternal celltraffic during pregnancy has been identified in healthy adults and inpatients with scleroderma (systemic sclerosis, “SSc”). HLA-specificprimers and fluorogenic probes were used in real-time quantitativepolymerase chain reaction assays to investigate the frequency andquantitative levels of maternal Mc (“MMc”) in healthy women and womenwith SSc.

Briefly, HLA-specific primers and probes were used to targetnon-inherited, non-shared HLA sequences. DNA-based HLA typing wasconducted in 67 proband mother pairs and in all children if the probandwas parous. Statistical analysis was limited to 50 proband mother pairs(including 32 healthy women and 18 women with SSc) in whom MMc could bedistinguished from potential fetal Mc. The probands were either healthywomen with no history of autoimmune disease, or women with scleroderma.A total of 253 subjects were studied and included healthy women (n=41)and their mothers (n=41) and children (n=58), and women with SSc (n=26)and their mothers (n=26) and children (n=39). The study population wasderived from an overall population of 74 healthy women and 56 women withSSc, among whom 85% had a non-inherited, non-shared maternal HLA-DRB1,DRB3, DRB4, DRB5, DQA1, DQB1, or B allele, with 70% of HLA differencesinformative using same panel of 8 HLA-specific primers in Q-PCR assaysas described in Example 1. Probands who were parous were included in thecurrent study only if all living children were also willing to bestudied. This requirement was included because fetal Mc from a priorpregnancy can confound the detection of MMc. Parity was similar in thetwo groups, and the study subjects were recruited from a similargeographic distribution (state of Washington and surrounding areas). Allsubjects provided informed consent.

Another potential source of microchimerism is blood transfusion (Lee etal., Blood 93:3127, 1999). The HLA primers used in this study arespecific for a particular HLA polymorphism, and chances are extremelylow that the blood donor had the same HLA allele as the mother of theproband. Of the 32 controls for whom information on blood transfusionhistory was available, one had had a blood transfusion prior to thestudy. Of the 25 scleroderma patients, five had had a blood transfusion(one prior to disease onset, one at the time of onset, and three afteronset).

Results. MMc in peripheral blood mononuclear cells was more frequentamong women with SSc (72%) than healthy women (22%) (odds ratio: womenwith SSc were 9.3 times more likely to have MMc than healthy women,P<0.001) (see Table 2).

TABLE 2 Frequency of MMc in Healthy Women and Women with SSc* No. (%)Odds P vs. Positive Ratio 95% CI Controls Model with all ProbandsControls  7/32 (21.9) 9.3 2.5-35 0.001 Cases 13/18 (72.2) Modelexcluding Probands with transfusions Controls  7/31 (22.6) 6.2 1.6-250.01 Cases  9/14 (64.3) *MMc = maternal microchimerism; SSc = systemicsclerosis; 95% CI = 95% confidence interval.

However, levels of MMc, expressed as the genome equivalent of maternalcells per million (gEq/mil), were not significantly different (0-68.6gEq/mil in SSc patients, 0-54.5 in healthy women) (see Table 3).

TABLE 3 Frequency of MMc in Healthy Women and Women with SSc* No. ofSubjects/ gEq/mil host cells, P vs. Observations median (range) ControlsModel with all Probands Controls 32/34 0 (0-54.5) 0.056 Cases 18/24 0.71(0-68.6) Model excluding Probands with transfusions Controls 31/33 0(0-54.5) 0.18 Cases 14/20 0.71 (0-34.5) *MMc = maternal microchimerism;SSc = systemic sclerosis; gEq/mil = genome equivalent per million.

In additional studies, positivity for MMc was demonstrated in a bonemarrow aspirate from an SSc patient in whom peripheral blood had beenfound to be negative for MMc on four occasions, and tissue from asubsequent autopsy of this patient had MMc levels of 757 and 1,489gEq/mil in the lung and heart, respectively (see FIG. 2 ).

Conclusion. MMc is not uncommon in the peripheral blood of healthyadults, is increased in frequency in patients with SSc, and may bepresent in bone marrow and disease-affected tissues even if absent inthe peripheral blood.

What is claimed is:
 1. A composition, comprising a forward nucleic acidmolecule, a reverse nucleic acid molecule, and a probe nucleic acidmolecule, wherein the forward, reverse and probe nucleic acid moleculesare complementary to SE-LR (DERAA (SEQ ID NO:89)), and wherein theforward nucleic acid molecule has a length of up to 23 nucleotides andcomprises the sequence as set forth in SEQ ID NO.:64, the reversenucleic acid molecule has a length of up to 26 nucleotides and comprisesthe sequence as set forth in SEQ ID NO.:65, and the probe nucleic acidmolecule comprises a nucleic acid sequence, a fluorophore and aquencher, wherein the nucleic acid sequence has a length of up to 18nucleotides and comprises the sequence as set forth in SEQ ID NO.:66. 2.The composition according to claim 1, wherein the fluorophore is locatedat the 5′-end of the probe nucleic acid molecule and the quencher islocated at the 3′-end of the probe nucleic acid molecule.
 3. Thecomposition of claim 2, wherein the fluorophore is FAM and the quencheris TAMRA or BHQ.
 4. The composition of claim 2, wherein at least onenucleotide of the probe is a duplex stabilizer.
 5. The composition ofclaim 4, wherein the duplex stabilizer is at least one LNA or MGB.
 6. Akit comprising a nucleic acid composition according to claim
 1. 7. Thekit of claim 6, further comprising one or more reagents for performingPCR.
 8. A process for detecting microchimerism, comprising: (a)amplifying target nucleic acid molecules of a test biological sampleusing one or more nucleic acid compositions according to claim 1,wherein the test biological sample comprises a known HLA genotype; (b)amplifying nucleic acid molecules of a control biological sample usingthe one or more nucleic acid molecule compositions of (a), wherein thecontrol biological sample comprises a known HLA genotype that isdifferent from the test biological sample; and (c) detecting thepresence of microchimerism when the amplification from the testbiological sample and the control biological sample identifies HLAmarkers in the test biological sample that are also present in thecontrol biological sample.
 9. The process of claim 8, wherein thedetection sensitivity is one chimeric genome in 10⁵ host genomes. 10.The process of claim 8, wherein the test biological sample is blood orserum.
 11. The process of claim 8, wherein the microchimerism detectedis maternal microchimerism.
 12. The process of claim 8, wherein themicrochimerism detected is fetal microchimerism.
 13. The process ofclaim 8, wherein amplifying comprises performing Q-PCR.
 14. A method fordetecting incipient allograft rejection, comprising: (a) obtaining afirst biological sample from a subject prior to receiving a transplantfrom a transplant donor and a second biological sample from the samesubject after receiving the transplant during a period of time whenthere is a risk of rejection; (b) amplifying target nucleic acidmolecules from the first and second biological samples using one or morenucleic acid compositions according to claim 1, wherein one or more ofthe nucleic acid compositions comprising the forward, the reverse, andthe probe nucleic acid molecules are complementary to target nucleicacid molecules found in the transplant donor and not found in thetransplant recipient; and (c) detecting incipient allograft rejection inthe transplant recipient when the presence of amplified target nucleicacid molecules from the transplant donor are detected in the transplantrecipient.
 15. The method of claim 14, wherein the first biologicalsample and/or the second biological sample is blood or serum.
 16. Themethod of claim 14, wherein the transplant comprises a kidneytransplant, a pancreas transplant, an islet cell transplant, ahematopoietic cell transplant, a cord blood transplant or a bone marrowtransplant.
 17. The method of claim 14, wherein the subject is human.18. The method of claim 14, wherein the subject has a known genotype.19. The method of claim 14, wherein amplifying comprises performingQ-PCR.